Decorated red blood cells

ABSTRACT

Disclosed are modified red blood cells which function as deployment platforms for important biomolecules. Such modified red blood cells can confer, for example, in vivo protection against exposure to an otherwise lethal nerve agent.

GOVERNMENT SUPPORT

This invention was made with Government Support under Contract NumberMDA972-96-K-0002 awarded by DARPA. The Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

The red blood cell is a dominant presence in the circulatory system,representing approximately 98% of the formed elements which are present.Therefore, these cells can be viewed as a potentially importantdeployment platform for a variety of biomolecules which can be attachedand displayed on the surface of the red blood cells. Barriers to someforms of such a deployment strategy include, for example, the fact thatsuch modified red blood cells may be short-lived in circulation, therebyrendering them less effective. A strategy for the successful developmentof a red blood cell platform could provide a means for the treatment/andor prevention of a wide range of human disorders.

SUMMARY OF THE INVENTION

The present invention relates to modified red blood cells which functionas deployment platforms for important biomolecules. Such modified redblood cells can confer, for example, in vivo protection against exposureto an otherwise lethal nerve agent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that red blood cells,modified as described herein, can function as a successful deploymentplatform for important biomolecules. More specifically, as discussed inthe Exemplification section which follows, Applicant has demonstrated invivo protection against exposure of an animal to an otherwise lethalnerve agent. Protection was provided by decorating red blood cells inthe animal with an enzyme capable of degrading the nerve agent.

Thus, the present invention relates, in one aspect, to a modified redblood cell which is relatively long-lived in circulation, the modifiedred blood cell bearing on its surface at least one biomolecule capableof neutralizing challenge by an endogenous or exogenous agent. As usedherein, the expression “long-lived in circulation” will be definedwithin the context of ex vivo modification and reintroduction. That is,when red blood cells are removed from an animal, modified ex vivo andreintroduced into the animal, the long-lived criteria is satisfied whenat least about 70% remain in circulation 24 hours after reintroduction.

The expression “biomolecule”, as used herein, refers to any moleculewhich may be found in a living organism. With respect to the presentinvention, proteins are the most significant class of biomolecules.However, other important classes of biomolecules are included within thescope of the present invention, including, for example, carbohydrates.In general, the role of the biomolecule on the surface of the red bloodcell is to either 1) act as an affinity reagent, specifically binding toanother biomolecule; or 2) act as a molecular tool, modifying ordegrading a biomolecule of interest.

As mentioned above, the proteins are a particularly significant class ofbiomolecules. The proteins include such important species as antibodies(which are useful as affinity reagents) and enzymes (which can catalyzethe modification or degradation of a biomolecule of interest).

As used herein, the expression “endogenous” refers to agents (e.g.,chemical or biological agents) which are typically found in the animalof interest. The expression “exogenous” refers to agents which are nottypically found in the organism of interest. Issues to be considered inconnection with the neutralization of endogenous versus exogenous agentsusing a biomolecule fixed to a red blood cell platform are notnecessarily identical. The experiments described in the Exemplificationsection which follows relate specifically to an exogenous chemicalagent.

Modified red blood cells of the type described herein are capable ofneutralizing challenge by an endogenous or exogenous agent. Theexpression “neutralizing challenge” can not be defined precisely for allendogenous or exogenous agents. Rather, one must consider eachendogenous or exogenous agent on a case by case basis, and consider theconsequences of exposure or challenge by such agents to determine themeaning of the term “neutralizing”.

Consider, for example, exogenous biological agents such as bacteria orviruses. Certain pathologies are associated with bacterial or viralinfection—such pathologies can be determined by reference to medicalhandbooks. “Neutralization”, as used herein, can refer, for example, tothe prevention, elimination, mitigation, or delay in onset of suchpathologies.

In the Exemplification section set forth below, an exogenous chemicalagent is considered. More specifically, a toxic nerve agent isintroduced into an animal carrying modified red blood cells of thepresent invention. In the absence of the modified red blood cells,animals exposed to the nerve agent die. Thus, in this context,“neutralization” refers to the fact that animals carrying the modifiedred blood cells survive.

An example which relates to an endogenous agent is LDL cholesterol. Itis known that LDL cholesterol is found in vivo in both oxidized andreduced states. It is the oxidized form of LDL cholesterol that isdangerous. It is ingested by the cells of an atherosclerotic plaquewhich swell causing occlusion. Certain individuals apparentlyunderexpress the enzyme responsible for maintaining LDL cholesterol inthe reduced form (glutathione peroxidase). A method of therapy in suchindividuals is to deploy this enzyme on the surface of red blood cellsthereby aiding in the maintenance of LDL cholesterol in the reducedform.

In another aspect, the present invention relates to a modified red bloodcell, the surface of which is decorated with an ensemble (i.e., aplurality) of biomolecules. Such an ensemble of molecules can work inconcert to achieve a desired neutralizing effect. The use of an ensembleof biomolecules is particularly important with respect to theneutralization of complex exogenous biological agents such as bacteriaand viruses.

For example, red blood cells can be modified to bear an antibody orantibodies specific for a bacterium of interest. Such antibodies canbring the modified red blood cell into contact with the bacterium ofinterest if present in the circulation system. Other biomoleculespresent on the surface of the red blood cell can be provided which havethe ability to breach the outer membrane/cell wall of the bacterium.These include, for example, lysozymes, bacteriocidal permeabilityincreasing peptides and other pore forming antimicrobials. In addition,the bacterial electron transport array may be used to generate hydroxylradicals within the bacterial inner cell membrane. Electron mediatorssuch as hemin, derivatives of quinones, menadione or methyl viologen maybe deployed on the surface of the red blood cell. Such electronmediators will produce hydroxyl radicals within the bacterial innermembrane by reducing oxygen directly. The penetration of such electronmediators will be assisted by the presence of lysozyme, which removesthe peptidoglycan and allows the interaction of the electron mediatorwith the inner membrane. Potential synergy with bacteriocidalpermeability increasing peptides for further disruption oflipo-polysaccharide or peptidoglycan layers is also likely.

The killing of bacteria by the addition of hemin has been demonstratedin relevant experiments. More specifically, this has been demonstratedin B. subtilis as well as S. aureus and other gram positive bacteria.Oxygen was required for bacterial killing. Bacteriocidal quantities ofhemin did not damage bacteria in the absence of oxygen. Porphyrinwithout iron was also tested and a lack of bacteriocidal effect wasobserved due to the essential role of Fe in electron mediation.Moreover, when Zn was substituted for Fe the resulting complexdemonstrated the expected reduction in bacteriocidal efficacy. It wasalso demonstrated that hemin, attached to polyethylene glycol tethers,does not kill bacteria with an intact peptidoglycan coat. The killing ofgram negatives was achieved with hemin, provided that thelipopolysaccharide layer was first disrupted with polyethylene imine.

Deploying and ordering the bioengineered macromolecules into amulticomponent array yields large functional dividends. It can readilybe demonstrated that a progression from unconnected to connected andordered elements leads to increasing efficacy. This can be demonstratedthrough the production of random attachments to red cells followed by aprogression to specific ordered attachments. The savings in diffusiontime and gains in substrate concentrations that arise from ordering sucha system are significant. Two principal technologies exist: a sequentialmethodology (such as is required for the use of most linkage strategiessuch as avidin-biotin and solid phase peptide synthesis) and a massivelyparallel, simultaneously self-assembling system (such as is possiblewith coded PNA constructions). The self-assembling PNA constructs willreliably preserve the topology that has been initially designed into thearray. The PNA strategies offer an advantage in that mild reactionconditions are required, high affinity and high specific binding isachieved and a virtually unlimited library of complementary sequencesare available.

More generally, biomolecules can be attached to the surface of red bloodcells in vitro using any appropriate chemical functionality. Forexample, PNAs linked to an activated carboxylic acid moiety via aprimary amino group represents one approach. Alternatively, attachmentof an avidin anchor on biotinylated red blood cells can be used toattach a biotinylated enzyme. The attachment of a biotin anchor on a redblood cell attached to an enzyme to which avidin has been linked is alsoan option. Finally, the use of tannin to anchor avidin to the red bloodcell platform for subsequent attachment of a biotinylated enzyme is alsoa viable option.

To create an optimal, stable foundation for the biomolecule ensemble, itmay be necessary to introduce sites on biomolecules which facilitateattachment to red blood cells. Chemical modification of natural proteinsis inexpensive and technically simple, but rarely permits site- andquantity-controlled reactions. Moreover, it never allows construction ata specified position on the protein surface that has been chosen by suchcriteria as orientation with respect to the substrate or to othercomponents of the ensemble. Alternatively, standard recombinanttechnology permits the facile engineering of special properties atspecific sites. These properties may be expressed by amino acid residueswith unusual chemistry, such as cysteine, cassettes that encodespecific, high-affinity binding domains, such as that for biotin, orsequences that direct specific enzymatic modification such as fatty acidconjugation.

Additional advantage can be gained by introducing attachment sites onbiomolecules. These sites allow the ensemble components to be readilyassembled into ordered arrays. The description in the precedingparagraph applies to sites required for the attachment of components tothe red blood cell surface. With a complex, highly organized ensemblecomes the need to engineer into a given component more than one site,each having its own special chemistry.

The chemistries for attaching PNA to these sites could havecommonalities, but site selection for PNA attachment would have to bemade on an enzyme-specific basis. Minor imprecision is tolerable if theprocess of self-assembly severely limits the incorporation of“incorrectly” modified components.

Using techniques such as those described above, 5,000 to 10,000 alkalinephosphatase molecules have been attached to various human and animalmodel red blood cells. The morphology, in vitro biophysical diagnosticsand in vivo persistence studies have been carried out. Avidin has beenmodified to add carbohydrate moieties to reduce undesirable hydrophobicinteractions on the avidin surface. A specific panel of in vitrobiophysical diagnostic tests for the prediction of human red cellsurvival in vivo have been developed. Advanced nano-fabricated arrayswhich simulate the properties of in vivo capillary channels have beendeveloped in order to evaluate the biophysical properties of decoratedcells. Biochemical methods have been developed in the form of sialicacid attachments for rendering enzymatic decorations invisible to theclotting and immune systems.

Enhanced catalysis and enhanced enzyme stability are also issuesrelevant to red cell deployment. Gains in specificity and efficiencyover those exhibited by wild-type enzymes may greatly improve theeffectiveness of the deployment system. Methods of library constructionvia mutagenesis and phage display are well-known in the art. To identifyan enzyme having enhanced activity it is first necessary to establish anefficient screening method. Improvement in specificity is a qualitativeissue and may require the synthesis of special substrates for use inconnection with ELISA or other high throughput assay systems.Improvement in efficiency is quantitative and the assay must be simpleand precise.

To enhance enzyme stability without following the experimental routeoutlined in the preceding paragraph, it may be useful to screenhigh-temperature microbes for a more stable version of an enzyme ofinterest. It may also be demonstrated that the incorporation of amarginally stable enzyme into a well-ordered ensemble will confer amicroenvironment which enhances stability.

Another advance in the implementation process is represented in thedevelopment of a new process for storing red blood cells which yieldsexcellent levels of recovery with in vivo 24 hour post-transfusionmeasurements after 9 weeks of storage.

In another aspect, the present invention relates to a method foroccluding the capillaries that feed inflammation and neoplasticprocesses, thereby eliminating, or reducing, associated pathologies. Itis known that tumors and inflammatory processes induce the formation ofnew capillary vessels which provide perfusion. These newly formedvessels are enriched in cell-adhesion molecules, relative to theirpre-existing counterparts in the body. By deploying red blood cellsbearing biomolecules which specifically bind to cell adhesion molecules,it is possible to specifically occlude the vessels which perfuse tumorsor inflammatory processes.

In addition to the ex vivo modification of red blood cells, in vivomodification is also possible. This would entail initial infusion ofanchoring molecules which primarily insert into red cells. A secondaryinfusion would set into place the designated biomolecular tool.

EXEMPLIFICATION

i) Biotinylation Procedure

Fresh rat blood was obtained through either cardiac puncture orvenipuncture of the subclavian vein. The cells were suspended to Hct 10in TEA buffer (50 mM triethanolamine, 100 mM NaCl, 10 mM glucose, 2 mMMgCl₂, adjusted to pH 7.9). NHS-biotin (Pierce catalog #21217) solution(1 mg/ml in 140 mM NaCl) was prepared. Cells were added to 0.03 mg/mlfinal concentration. The suspended cells were then incubated at roomtemperature for 30 minutes on a Nutator.

Following incubation, the cells were washed once in ALP (128 mM NaCl, 10mM glucose, 10 mM Na HEPES, 1 mM CaCl₂, 0.5 mM MgCl₂, adjusted to pH7.4) buffer with 10 mg/ml bovine serum albumin (BSA). The cells werethen resuspended to Hct 10 in ALP-BSA (BSA 10 mg/ml) buffer. Neutravidin(Pierce catalog #31000) 1 mg/ml solution in ALP-BSA (BSA 10 mg/ml)buffer was added to cells in a 1:10 ratio (i.e., to 500 ul cells, 50 ulneutravidin solution was added). The cells were incubated at roomtemperature for 30 minutes on a Nutator. The cells were then washed oncewith ALP-BSA (BSA 1 mg/ml) buffer.

The cells were then resuspended to Hct 10 in ALP-BSA (BSA 1 mg/ml)buffer. Biotinylated paraoxonase was added to cells in a saturatingamount (assuming a level of decoration of approximately 20,000/cell).The cells were incubated at room temperature for 30 minutes on aNutator. The incubated cells were then washed with ALP-BSA (BSA 1 mg/ml)buffer. The number of decorations/cell was determined and cells wereprepared for injection.

ii) Injection Protocol

The cells to be injected were prepared in a volume of approximately 10%of the animal's blood volume. In the rats of the present experiment thiswas calculated as 70 ml blood/kg body weight. The rats were anesthetizedusing a mixture of ketamine (95 mg/kg) and xylezine (12 mg/kg). Atourniquet was applied to the animal's tail and a catheter was insertedinto one of the lateral tail veins. The preparation of decorated cellswas injected slowly. Approximately 5 minutes after injection, a bloodsample was obtained though a subclavian venipuncture to assess thesuccess of the injection. The animal was allowed to recover prior tochallenge.

iii) Results

Dosages of paraoxone were administered i.p. to 170 g Fisher rats. 5Xparaoxone (X=published LD50) was uniformly lethal in control rats havingno modified red blood cells. In the experimental rat population, themodified red blood cells were fully protective to challenge at 5X (2 outof 2 rats), 7X (3 out of 3 rats) and 10X (2 out of 2 rats) paraoxone.

1. A method for preventing the death of an individual resulting fromexposure to a toxic chemical agent, comprising providing red blood cellsin the circulatory system of the individual, the red blood cells beingdecorated with an enzyme which destroys the toxic chemical agent, andsubsequent to decoration being reintroduced into the individual.
 2. Themethod of claim 1 wherein the decorated red blood cells are reintroducedat a level of between about 5% to about 20% of the total red blood cellpopulation.
 3. The method of claim 1 wherein the toxic chemical agent isa nerve agent.
 4. The method of claim 3 wherein the nerve agent causesdeath in an individual by inhibiting synaptic acetylcholinesterase. 5.The method of claim 4 wherein the nerve agent is an organophosphoruscholinesterase inhibitor.
 6. The method of claim 5 wherein the nerveagent is a G-agent.
 7. The method of claim 6 wherein the G-agent isselected from the group consisting of GB, GD and GF.
 8. The method ofclaim 5 wherein the nerve agent is VX.